1. Field of the Invention
This invention relates generally to the field of fluorescence spectroscopy. More particularly, this invention relates to an improved technique and apparatus for making fluorescence measurements with increased sensitivity.
2. Description of Related Art
Fluorescence spectroscopy is a widely used technique for performing analytical studies, both quantitative and qualitative, of organic and inorganic materials. Spectroscopic studies of fluorescence characteristics of materials when subjected to radiation of certain wavelengths constitute a convenient means for obtaining information regarding material composition, purity, concentration, turbidity, etc.
In its basic form fluorescence spectroscopy involves the examination of fluorescent radiation resulting from exposing the medium or sample under scrutiny to exciting radiation. In making fluorescence measurements, the sample, which is usually in the form of a solution or a suspension of solid particles in a liquid, is placed within a beam of the desired exciting radiation. The exciting radiation causes the sample to fluoresce and the resulting fluorescence is sensed by a detector and examined. The emitted radiation is usually filtered by some form of secondary wavelength filters or mono- chromators in order to achieve maximum separation of the exciting radiation from the emitted fluorescence, prior to being detected by a photodetector.
Integrity of fluorescence measurements is directly related to the degree to which fluorescent radiation emanating from a sample is isolated from the exciting radiation originally incident upon the sample. Further, the sensitivity of measurement is a function of the extent to which the emitted radiation is actually detected by the photodetector. Achieving these objectives to an optimum extent constitutes a difficult obstacle for conventional fluorescence spectrometry systems and has significantly restricted their operational efficiency.
Problems associated with separation of excited radiation from incident radiation and with detecting a major segment of excited radiation are further compounded when the material or sample under scrutiny is in liquid form. In conventional systems, fluorescence measurements of liquid samples are carried out in standard glass or fused silica cuvettes with exciting radiation or light being focused onto the sample contained within the cuvette through one side of the cuvette. Radiation emitted as the sample fluoresces is measured at right angles to the exciting light.
In such systems, monochromators or interference filters formed of glass or fused silica are used to separate the exciting light from the emission light. This separation is an important aspect of the measurement process and is necessary because the exciting light which is scattered by the sample under scrutiny is typically of much higher intensity than the intensity of the fluorescent emission from the sample. The problem is further compounded by the fact that the photodetectors used for measuring the fluorescent emission generally respond to the exciting wavelengths as well.
Under these conditions, the optics arrangements used with conventional systems for fluorescence measurements are capable of collecting and detecting only a limited amount of emission light from the test cuvette. Since the excitation light is directed into one side of the cuvette while the resulting fluorescence is emitted at equal intensity in all directions, the amount of light which can be gathered is inherently a small fraction of the total amount emitted. Accordingly, the signal-to-noise ratio achievable by conventional fluorescence systems is restricted, thereby restricting the selectivity, efficiency and sensitivity with which fluorescent spectroscopic studies can be made.
Various types of optics arrangements have been used in conventional fluorometry systems in an attempt to improve the efficiency with which fluorescent light emitted from a test sample is collected. Reflective optics have been advantageously used in this regard. As an example, reflective optical components have been employed which surround the conventional glass or fused silica fluorescence cuvettes. Such reflective components surround the cuvette optically and increase coupling of fluorescent emissions to the conventional optics means for collecting emission light. Such arrangements still fall short of being optimum because the reflective optics require transmitting the fluorescent light back through the sample and the cuvette, thereby attenuating the intensity of emitted light considerably.
An additional problem associated with fluorescence measurements in cuvettes results from surface effects which arise regardless of whether the cuvette is composed of glass, fused silica or even plastic. The problem arises because some materials are capable of being selectively adsorbed on the cuvette surfaces. The absorption generates an increase in the fluorescent background level since the exciting light passes through and excites fluorescence from the adsorbed layer prior to reaching the sample solution which is to be excited. In addition, the adsorbed layer also attenuates the intensity of exciting light, thereby reducing the intensity of light available for exciting fluorescence from the sample. The end result is that signal-to-noise and signal-to-background ratios are substantially less than optimum with conventional fluorescence measurement techniques.